Throughout this application various publications are referred to in parentheses by number. Full citations for these references may be found at the end of the specification. The disclosures of each of these publications, and of all patents, patent application publications and books cited herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
The liver occupies a central position in life due to its crucial metabolic, synthetic, storage, and drug or toxin disposal functions. Isolated liver cells are extremely useful for developing disease models, as well as for toxicological testing and drug development. Moreover, because many proteins are made in liver cells, cell/gene therapy directed at the liver is of extensive interest for a long list of genetic or acquired conditions. However, shortages of donor organs have proved to be an insurmountable hurdle for therapeutic and other applications of liver cells. Therefore, alternative means to generate hepatocytes, e.g., from pluripotent stem cells, is of great interest. This requires understanding into the processes by which pluripotent stem cells may transition and differentiate, first into immature and then into mature hepatocytes. Among candidate pluripotent stem cells of interest, human embryonic stem cells (hESC) or induced pluripotent stem cells (iPS), which share properties of the former, divide indefinitely and may differentiate to produce mature cells of various tissues and organs. However, available differentiation protocols to generate hepatocytes from hESC or iPS, etc., are inefficient and generate cells of indeterminate developmental or maturational stages. For instance, the convention of generating hepatocytes from aggregation of hESC or other types of pluripotent stem cells to form embryoid bodies is not only inefficient, but yields complex lineage mixtures at various developmental stages or maturity that pose difficulties in isolating cells of interest, which may be additionally altered or damaged by cell separation procedures (1). Directed differentiation of stem cells into hepatocytes could overcome these problems, (2, 3), but this accomplishment has generally been elusive.
The present invention addresses the need for directed differentiation of stem cells into hepatocytes.